Methods for identifying alpha PDGFR agonists and antagonists

ABSTRACT

Discoveries are disclosed that show particular aspects of recombinant DNA technology can be used successfully to produce a hitherto unknown type of human Platelet-Derived Growth Factor (PDGF) receptor protein free of other PDGF receptors. These proteins can be produced from DNA segments in cells in various functional forms. These forms variously enable biochemical and functional studies of these novel receptors as well as production of antibodies. Means are described for determining the level of expression of genes for specific types of PDGF receptor proteins, for example, by measuring mRNA in cells with PDGF receptor type-specific DNA probes or by measuring antigen in biological samples with type-specific antibodies.

This application is a continuation of Ser. No. 09/769,987, filed Jan.25, 2001, now U.S. Pat. No. 6,660,488 (14014.0266U2), now allowed, whichis a divisional of Ser. No. 08/460,656, filed Jun. 2, 1995(14014.0266US), now U.S. Pat. No. 6,228,600, issued May 8, 2001, whichis a divisional of 08/439,095, filed May 11, 1995 (14014.0279), pending,which is a continuation of Ser. No. 07/915,884, filed Jul. 20, 1992,abandoned, which is a continuation of Ser. No. 07/308,282, filed Feb. 9,1989, abandoned. All of these applications are herein incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to genes which encode receptor proteinsfor Platelet Derived Growth Factor (PDGF), particularly to those humangenes encoding receptor proteins which preferentially bind the majorform of human PDGF which is found in platelets. This invention alsorelates to synthesis of products of such PDGF receptor genes byrecombinant cells, and to the manufacture and use of certain other novelproducts enabled by the identification and cloning of DNAs encodingthese receptors.

BACKGROUND OF THE INVENTION

Genes encoding growth factors and their receptors have been implicatedin the regulation of normal cell growth and development. There is alsoincreasing evidence that genetic alterations affecting expression ofsuch genes can contribute to altered cell growth associated withmalignancy. The normal homologues of some oncogenes code formembrane-spanning growth factor receptors with tyrosine kinase activity(2, 3). Other oncogenes appear to act in pathways of growth factoractivated cell proliferation as well (4). Thus, increased knowledge ofgrowth factor regulatory systems in general is expected to providebetter understanding of genes critically involved in both normal growthcontrol and neoplasia.

Platelet-Derived Growth Factor (PDGF) is of particular importancebecause it is a major connective tissue cell mitogen which is thought toplay a major role in normal wound healing. Further, the abnormalexpression of PDGF has been implicated not only in cancers, but also ina variety of histopathologic states including arteriosclerosis,arthritis, and fibrotic diseases (23).

PDGF consists of a disulfide-linked dimer of two polypeptide chains,designated A and B. There is evidence for the natural occurrence of allthree possible dimeric structures containing A or B chains or both (1,25, 26). The various dimeric forms of the growth factor are called“isoforms”. A variety of normal and neoplastic cells appear tospecifically express either the A or B chains. Nevertheless, the mostsignificant human isoform for physiological regulatory processes isbelieved to be the one isolated from human platelets, namely the ABheterodimer (i.e., a dimer containing one A and one B chain; seereference 24).

The PDGF-A and B chains have distinguishable properties (37). The Achain is much more efficiently secreted and exhibits lower specificmitogenic activity than the B chain. The B chain gene of PDGF has beenshown to be the normal human homoloque of the simian sarcomavirus-derived v-sis oncogene. Moreover, there is accumulating evidencethat expression of the B chain in cell types possessing PDGF receptorscan drive such cells along the pathway to malignancy. The A chain isless potent than the B chain in inducing neoplastic transformation ofcultured mouse (NIH/3T3) cells.

Recent studies have suggested the existence of two subtypes of the PDGFreceptor (PDGF-R), on the basis of PBGF isoform binding and competitionusing mouse or human fibroblasts (27). These works are consistent withthe hypothesis that there exists one receptor subtype whichpreferentially binds the B chain dimer, and another which efficientlybinds all isoforms of the PDGF molecule. However, the results of thesestudies could not discriminate between two distinct possibilities withdiffering implications for the study and ultimate treatment of diseasesinvolving such receptors: either these subtypes represent differentlyprocessed products of a single PDGF-R gene; or they are products ofdistinct genes.

Further, there have been conflicting findings concerning binding ofdifferent PDGF isoforms of the receptor produced by a previouslyidentified human PDGF-R gene. Introduction of PDGF-R genes by expressionvectors into different cell types devoid of PDGF receptors has beenreported to lead either to preferential binding of PDGF-BB (14) or,alternatively, to efficient binding by all three isoforms (28). Thebasis of this discrepancy is not known.

Thus, there has been uncertainty concerning the ability of the knownPDGF receptor to respond to different PDGF isoforms, and to the main ABheterodimer form of human PDGF, in particular. Some reported differencesmight be explained by cell specific differences in post-translationalprocessing of the product of the known PDGF-R gene, or by the presenceof accessory proteins in certain cell types. Alternatively, thedifferent binding properties reported in different studies might beexplained by the existence of two distinct genes encoding different PDGFreceptors.

In light of the complexities of PDGF ligand and receptor activitiesdescribed above, and the related processes which are influenced thereby,comprising both normal wound healing and abnormal connective tissueconditions, including neoplastic growth, arteriosclerosis, arthritis,and fibrotic diseases, it is apparent that there has been a need formethods and compositions and bioassays which would provide an improvedknowledge and analysis of mechanisms of connective tissue growthregulation, and, ultimately, a need for novel diagnostics and therapiesbased on the PDGF receptors involved therein.

In particular, the observations above indicate a specific need forthorough characterization of the genetic basis of PDGF receptorproduction. Furthermore, it has been shown previously (5) that it ispossible to identify and clone novel related members of the gene familyencoding membrane-spanning growth factor receptors with tyrosine kinaseactivity, which comprises the known PDGF receptor gene and the kit andfms oncogenes, by exploiting the conserved tyrosine kinase coding regionas a probe.

Accordingly, the present invention contemplates the application ofmethods of recombinant DNA technology to fulfill the above needs and todevelop means for producing PDGF receptor proteins which appear to bethe predominant effectors of the main form of human PDGF. This inventionalso contemplates the application of the molecular mechanisms of thesereceptors related to healing and pathological processes.

In particular, it is an object of the present invention to identify andisolate the coding sequence of a novel human gene related to butdistinct from the known PDGF-R gene, as well as from other members ofthe family of tyrosine kinase genes comprising the PDGF-R, kit, and fmsgenes. Further, it is an object of this invention to develop themolecular tools needed to establish the relative roles of the novel andknown forms of PDGF receptor in physiological processes involving PDGF.

SUMMARY OF THE INVENTION

The present invention relates to a development of recombinant DNAtechnology, which includes production of novel PDGF receptor (PDGF-R)proteins, free of other peptide factors. Novel DNA segments, RNAs, andbioassay methods are also included.

The present invention in particular relates, in part, to DNA segmentswhich encode messenger RNAs (mRNAs) and proteins having structuraland/or functional characteristics of a new human receptor within thesubfamily of membrane-spanning tyrosine kinase receptor genes comprisingthe following known receptor genes: the PDGF-R gene; colony stimulatingfactor one receptor (CSF1-R) gene (also known as a cellular form of thefms oncogene, c-fms); and a cellular form of the kit oncogene (c-kit)(see references 3, 6, and 7 for background).

More specifically, this invention includes DNA segments containing agenomic DNA sequence or a DNA sequence complementary to the mRNAtranscribed from said genomic DNA (i.e., a “cDNA”), with a predictedprotein product similar in structure to other receptors of this growthfactor receptor subfamily. Among these receptors, the predicted novelgene product exhibits closest sequence homology to the known DGFreceptor.

Further, this novel product encoded by DNAs of this invention iscoexpressed with the known PDGF receptor gene product in a variety ofnormal cell types. This protein product can bind to and be functionallyactivated by PDGF. However, the activities of different PDGF isoformsfunctionally distinguish the new product, herein designated the type αhuman PDGF receptor, from that of previously identified genes encodingreceptors that can bind PDGF, including the known receptor previouslycalled the PDGF receptor and herein designated as the type β PDGFreceptor. Moreover, considerable evidence disclosed herein indicatesthat this novel gene product, the type α PDGF receptor, is the maineffector of activity for the most abundant form of PDGF in the humanbody.

In the practice of one embodiment of this invention, the DNA segmentsare capable of being expressed in suitable host cells, thereby producingthe novel PDGF receptor proteins. This invention also relates to mRNAsproduced as the result of transcription of the sense strands of the DNAsegments of this invention. The invention further comprises novelbioassay methods for determining levels of expression in human cells ofthe mRNAs and proteins produced from the genes related to DNA segmentsof the invention.

In a principal embodiment, the present invention comprises DNA segmentsencoding novel PDGF receptors, as exemplified by the following: a cloneof genomic normal human thymus DNA, herein designated as the T11 genomicclone; human cDNA clones of cell mRNAs containing sequences contained inT11, designated HF1, HB6, EF17 and TR4; and related DNA segments whichcan be detected by hybridization to any of the above human DNA segments,which related segments encode receptor genes, wherein said genes do notinclude previously known PDGF-related receptor genes.

The human gene related to clone T11 are referred to hereinafter as “theT11 gene” and use of the term “T11” as an adjective is intended toinclude any of the above DNA segments of this invention, absent aspecific reference to “the T11 genomic clone”.

In another embodiment, this invention relates to a recombinant DNAmolecule comprising a vector and a DNA of the present invention. Theserecombinant molecules are exemplified by molecules comprising genomic orcDNA clones related to the T11 gene and any of the following vector DNAsa bacteriophage λ cloning vector; or an expression vector capable ofexpressing inserted DNAs in mammalian cells.

In still another embodiment, the invention comprises a cell, preferablya mammalian cell, transformed with a DNA of the invention. Further, theinvention comprises cells, including yeast cells and bacterial cellssuch as those of E. coli and B. subtilis, transformed with DNAs of theinvention. According to another embodiment of the invention, thetransforming DNA is capable of being expressed in the cell, therebyincreasing the amount of PDGF-R protein encoded by this DNA, in thecell.

Still further, the invention comprises novel PDGF-R proteins made byexpression of a DNA of the invention, or by translation of an RNA of theinvention. These receptors can be used for functional studies, and canbe purified for additional biochemical and functional analyses, such asqualitative and quantitative receptor binding assays. In particular,these type α PDGF receptors may be used for the development of therapiesfor conditions involving abnormal processes involving PDGF and itsreceptors, by testing receptor binding and activation activities ofpotential analogs (either antagonists or agonists) of the various PDGFisoforms, including the main form of human PDGF.

According to this aspect of the invention, the novel PDGF-R proteins canbe protein products of “unmodified” DNAs and mRNAs of the invention, orthey can be modified or genetically engineered protein products. As aresult of engineered mutations in the DNA sequences, modified PDGF-Rproteins have one or more differences in amino acid sequence from thecorresponding naturally occurring “wild-type” proteins. Thesedifferences may impart functional differences to the modified geneproducts such as improvements in their manufacturability or suitabilityfor use in bioassays.

This invention also relates to novel bioassay methods for detecting theexpression of genes related to DNAs of the invention. According to onesuch embodiment, DNAs of this invention, particularly the most preferredDNAs, may be used as probes to determine specific levels of mRNAsrelated to type α PDGF receptors, without interference from mRNAs ofknown PDGF receptor genes. Such bioassays may be useful, for example,for identification of various classes of tumor cells or of geneticdefects in connective tissue growth and/or the healing response.

This invention further comprises novel antibodies made against a peptideencoded by a DNA segment of the invention or by a related DNA. In thisembodiment of the invention, the antibodies are monoclonal or polyclonalin origin, and are generated using PDGF receptor-related polypeptidesfrom natural, recombinant or synthetic chemistry sources. Theseantibodies specifically bind to a PDGF-R protein which includes thesequence of such polypeptide. Preferably, these antibodies bind only totype α PDGF receptor proteins or, alternatively, only to type α PDGFreceptor proteins. Also, preferred antibodies of this invention bind toa PDGF receptor protein when that protein is in its native (biologicallyactive) conformation.

Fragments of antibodies of this invention, such as Fab or F(ab)′fragments, which retain antigen binding activity and can be prepared bymethods well known in the art, also fall within the scope of the presentinvention. Further, this invention comprises pharmaceutical compositionsof the antibodies of this invention, or active fragments thereof, whichcan be prepared using materials and methods for preparing pharmaceuticalcompositions for administration of polypeptides that are well known inthe art and can be adapted readily for administration of the presentantibodies without undue experimentation.

These antibodies, and active fragments thereof, can be used, forexample, for specific detection or purification of either the novel typeα PDGF receptor, or, alternatively, of the known type β PDGF receptor.Such antibodies could also be used in various methods known in the artfor targeting drugs to tissues with high levels of PDGF receptors, forexample, in the treatment of appropriate tumors with conjugates of suchantibodies and cell killing agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Detection of v-fms and PDGF receptor related gene fragments inhuman placenta and thymus DNAs. Hybridization of a v-fms probe (A) or amouse PDGF receptor probe (B) to human placenta (lane 1 and 3) or thymus(lane 2 and 4) DNAs under stringent (50% formamide; lane 1 and 2) orrelaxed (30% formamide; lane 3 and 4) hybridization conditions. Arrowsindicate the 12-kbp EcoRI fragment detected under relaxed conditions byboth v-fms and mouse PDGF-R probes. FIG. 1 illustrates detection of genefragments related to the oncogene v-fms and to the known mouse PDGFreceptor in human placenta and thymus DNAs by Southern blothybridization analyses.

FIG. 2 Molecular cloning of the λT11 genomic fragment as well as cDNAsof T11 and PDGF-R genes. Restriction map of: λT11 genomic clone (solidlines); T11 cDNA clones (solid bars); and PDGF-R cDNA clones (openbars). Coding regions within three fragments, as determined bynucleotide sequencing analysis, are indicated by black boxes labeled a,b and c. FIG. 2 presents the restriction map of the novel v-fms-relatedgene (T11) and related human PDGF receptor cDNA clones.

FIG. 3 T11 cDNA nucleotide and predicted amino acid sequences.Nucleotides are numbered at the left. The predicted amino acid sequenceof the long open reading frame is shown above the nucleotide sequence.Amino acids are numbered over the amino acids, starting at the putativeinitiation codon. The potential N-terminal signal sequence isunderlined. Potential sites of N-linked glycosylation are overlined, andcysteine residues are boxed. The putative single transmembrane region isindicated by a shaded bar. The potential ATP binding site in the kinasedomain is indicated by circles over Gly at residues 600, 602 and 605 andLys at residue 627. The putative tyrosine autophosphorylation site atresidue 849 is indicated by *. The regions of the λT11 genomic sequencedefined by exons a, b and c are underlined. The AATAAA box close to thepolyadenylated 3′ end of the cDNA is underlined as well. FIG. 3 containsthe nucleotide sequence and deduced amino acid sequence of the noveltype α PDGF receptor encoded by the T11 gene.

FIG. 4 Hydropathicity profile and homology with other tyrosine kinasesof the T11 receptor like gene product. A schematic diagram of thepredicted protein domains shows the signal sequence (S; black box),ligand binding domain (LB), transmembrane domain (TM; second black box),juxtamembrane domain (JM), tyrosine kinase domains (TK1, TK2; hatchedboxes), inter-kinase domain (IK) and carboxyl terminus (C). Thehydropathicity profile was calculated by the method of Kyte andDoolittle (46). The homology percentages shown refer to identical aminoacids within each respective domain. Abbreviations: IR, insulinreceptor; EGF-R, epidermal growth factor receptor; ND, not determined.FIG. 4 depicts results of hydrophobicity analysis of human type α PDGFreceptor and homologies of deduced amino acid sequences in comparisonwith the known type β PDGF receptor and other receptors.

FIG. 5 Chromosome mapping of the T11 gene.

(A) Distribution of silver grains on normal human chromosomes by in situhybridization with pT11-P probe (clone of the 3.6-kbp PstI genomicfragment) (see FIG. 1). (B) Distribution of grains on chromosome 4. FIG.5 shows chromosome mapping of the type α PDGF receptor gene.

FIG. 6 Comparison of mRNA species of the T11 and known PDGF-R genes, innormal and tumor cells. The same filter was first hybridized with theprobe from pT11-HP (0.95-kbp HindIII-PstI genomic fragment) (A) and thenrehybridized with a PDGF-R cDNA probe (B). A different filter was firsthybridized with T11 cDNA (3.5-kbp BamHI fragment of TR4 including thewhole coding region) (C) and then rehybridized with PDGF-R cDNA (3.8-kbpNdeI fragment of HPR2) (D). A and B contained poly (A)+ RNAs (5: μg perlane) extracted from human smooth muscle (lane 1), heart (lane 2), liver(lane 3), spleen (lane 4) or embryo (lanes 5 and 6). C and D containedtotal RNA (20: μg per lane) extracted from G402 leiomyoblastoma cells(lane 1), SK-LMS-1 leiomyosarcoma cells (lane 2), A1186 or A204rhabdomyosarcoma cells (lanes 3 and 4), 8387 fibrosarcoma cells (lane5), astrocytoma tissues (lanes 6 and 7), A1690 astrocytoma cells (lane8), A1207 or A172 glioblastoma cells (lanes 9 and 10) or A875 melanomacells (lane 11). Migrations of 28S and 18S ribosomal RNA (markers) areas indicated. FIG. 6 is a comparison of mRNA species produced from thetype α and β PDGF receptor genes.

FIG. 7 Detection of T11 and PDGF-R proteins with peptide antisera inhuman cell lines (A) and COS-1 cell transfectants (B). (A) M426 humanembryo fibroblasts (lanes 1, 4,7 and 10), 8387 fibrosarcoma cells (lanes2, 5, 8 and 11), A204 rhabdomyosarcoma cells (lanes 3, 6, 9 and 12), (B)COS-1 cells (lanes 1 and 4), COS-1 cells transfected with vectorscarrying T11 cDNA (lanes 2 and 3) or PDGF-R cDNA (lanes 5 and 6). FIG. 7demonstrates specific detection of type α or type β proteins withpeptide antisera in human cell lines or in monkey (COS-1) cellstransformed with a T11 DNA in an expression vector.

FIG. 8 Binding of ¹²⁵I-labeled human PDGF to mouse control NIH/3T3,control COS-1 and COS-1 cells transfected with T11 or known PDGF-R cDNAexpression vectors. Results represent the mean values (±SD), oftriplicate samples. FIG. 8 displays binding Of (¹²⁵ I-labeled) humanPDGF to mouse cells (NIH/3T3), control COS-1 cells and COS-1 cellstransformed with T11 or known PDGF-R cDNA expression vectors.

FIG. 9 Tyrosine autophosphorylation of type α and type β PDGF-R geneproducts induced by different PDGF isoforms. A204 (A), 8387 (B), orNIH/3T3 (C) cells were incubated with PDGF-BB (30 ng/ml) (lane 2), humanPDGF (30 ng/ml) (lane 3), PDGF-AA (300 ng/ml) (lane 4) or 3 mM aceticacid (vehicle control: lane 1). Cell lysates were immunoprecipitatedwith peptide antisera directed against predicted type α or type β PDGFreceptors (anti-T11 and anti-HPR, respectively). Immunoblot analyses waswith antibodies to the receptors or phosphotyrosine (anti-P-Tyr) (54) asindicated above the blots. Arrows indicate the specific bands which wereblocked in the presence of immunizing peptide. FIG. 9 demonstratestyrosine autophosphorylation of type α and type β PDGF receptors inresponse to various isoforms of PDGF.

FIG. 10 Stimulation of DNA synthesis by PDGF-AB (triangles) or PDGF-BB(circles) in various cells, as follows: (A) mouse NIH/3T3; (B) humanM426; (C) human AG1523; (D) human M413. FIG. 10 shows preferentialstimulation of DNA synthesis by PDGF isoform, AB in various cells withhigher levels of type α PDGF receptor than type β receptor.

FIG. 11 Receptor binding of PDGF-AB (triangles) or PDGF-BB (circles) byhuman D32 cells reconstituted with type α (open symbols) or type β(filled symbols) PDGF receptors by transfection with vectors bearing therespective cDNAs. The inset displays the same data replotted in thestandard (semi-log) Scatchard format. FIG. 11 presents binding data fortype α and type β PDGF receptors on (human 32D) cells transfected withvectors bearing the respective cDNAs, demonstrating that the type βreceptor shows a strikingly lower affinity for the PDGF-AB form.

FIG. 12 DNA synthesis stimulation responses to PDGF-AB (triangles) orPDGF-BB (circles) by human D32 cells reconstituted with type α (upperpanel) or type β (lower panel) PDGF receptors. FIG. 12 illustrates thesimilar mitogenic responses to PDGF-BB by cells containing either type αor type β PDGF-R and the significantly lesser DNA synthesis response toPDGF-AB in the type β, compared to type α receptor containing cells.

FIG. 13 Chemotaxic responses to PDGF-AB (triangles) or PDGF-BB (circles)by human D32 cells reconstituted with type α (upper panel) or type β(lower panel) PDGF receptors. FIG. 13 demonstrates equivalent chemotaxiccellular responses to PDGF-BB in cells with type α or β PDGF-R, whereasPDGF-AB elicited a considerably lower chemotaxic response with type βreceptors than with type α receptors.

FIG. 14 Responses of inositol phosphate formation and cytosolic calciumion mobilization (i.e., [Ca²]i; data in insets) to PDGF-AB (triangles)or PDGF-BB (circles) by human D32 cells reconstituted with type α (upperpanel) or type β (lower panel) PDGF receptors. FIG. 14 shows the effectof PDGF-AB and PDGF-BB on inositol phosphate formation and cytosoliccalcium mobilization ([Ca²+]i) in cells bearing type α and type βPDGF-R, with the type β receptors again responding more efficiently toPDGF-AB.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The DNAs of this invention are exemplified by DNAs referred to hereinas: the T11 genomic clone; and clones HF1, HB6, EF17 and TR4, comprisinghuman cDNA clones of cell mRNAs containing sequences included in the T11genomic clone.

The T11 genomic clone and the TR4 cDNA clone are preferred DNAs of thisinvention. A clone designated pT11-HP (a HindIII-PstI 0.95-kbp fragmentof genomic clone T11) and a particular restriction fragment from a T11cDNA (3.5-kbp BamHI fragment of TR4, including the whole coding region)are most preferred DNAs of this invention.

The restriction enzyme digestion maps of cDNA clones HF1, HB6, EF17 andTR4, and their mapping relationships to genomic clone T11, are displayedin FIG. 2. The sense strand DNA nucleotide sequence, and the predictedprimary protein sequence encoded, are shown in FIG. 3 for the TR4 cDNAclone, the largest cDNA clone related to the T11 gene.

As described in the Experimental Section, the T11 genomic clonecomprises a clone of genomic fragment of normal human thymus DNAcontaining a 12-kbp sequence bounded by recognition sites for therestriction enzyme EcoRI, which fragment hybridized more strongly inanalyses by blot hybridization than other fragments with DNA probesderived from the tyrosine-kinase domains of both the viral oncogenev-fms and the mouse cellular PDGF-R gene (see FIG. 1). The T11 genomicclone contains most of the blocks of sequences found in the mRNA productof the T11 gene (i.e., the exons), in addition to intervening genesequences not found in the mRNA (i.e., introns).

Other DNAs of this invention include the recombinant moleculescomprising T11-related genomic or cDNA clones of this invention and anyof the following vector DNAs: a bacteriophage λ cloning vector(exemplified by λEMBL4 or λgt11); or a mammalian expression vector (suchas the pSV2 gpt vector into which the simian sarcoma virus promoter wasengineered) capable of expressing inserted DNAs in mammalian (e.g.,COS-1) cells.

Genomic clone T11 DNA was isolated, by standard gene cloning methodswell known in the art, from a genomic library constructed fromEcoRI-digested normal human thymus DNA which was size-selected bysucrose gradients and cloned into the λEMBL-4 vector system. The λT11clone was identified on the basis of hybridization with both v-fms andmouse PDGF-R probes only under relaxed but not stringent hybridizationconditions. Further details of the cloning strategy and probes areprovided below and in the following Experimental Section.

A plasmid containing the HF1 cDNA clone, designated pHF1, was isolatedby standard, well known methods, from a normal human fibroblast cDNAlibrary in the Okayama-Berg expression vector under stringent conditionsusing the 0.9-kbp HindIII-PstI fragment of T11 which is a most preferredDNA of this invention. It contains a 3.9-kbp cDNA insert whichhybridized to a 6.4-kb RNA transcript in normal human fibroblasts andcontains a polyadenylation signal followed by a poly(A) tail at its 3′end. It also contains the coding sequence within the λT11 DNA and 170nucleotides related to CSF1-R and PDGF-R tyrosine kinase domainsupstream of exon (a).

The cDNA clone kHB6 was isolated by standard methods using the 0.4-kbp5′ end of clone HF1 to screen a human infant brain cDNA library in theλgt11 vector.

Another cDNA clone, λEF17, isolated by screening a human embryofibroblast (M426 cell line) cDNA library, prepared by random priming ofDNA synthesis on mRNA template and cloning in the λgt11 vector, with a0.2-kbp 5′ fragment of λHB6 as a probe. A possible ATG initiation codonwas identified within EF17.

The three overlapping clones (pHF1, λHB6 and λEF17) contain the entirecoding region in addition to 138-bp 5′ and 3-kbp of 3′ untranslatedsequences (FIG. 2).

The cDNA clone TR4 was obtained using a 5′ 0.2-kbp subfragment of λEF17to screen a M426 human embryo fibroblast cDNA library in a “phagemid”(phage and plasmid hybrid) vector (10). The 6.4-kbp TR4 cDNA cloneincludes an open reading frame beginning with a possible ATG initiationcodon at nucleotide position 139 and extended to a TAA termination codonat position 3406 (see FIG. 3). Moreover, the first 23 amino acid stretchdisplayed properties of a cleavable hydrophobic signal peptide (FIGS. 3& 4). The open reading frame was followed by 3-kbp of untranslatedsequences and a polyadenylation signal (AATAAA) located 25 nucleotidesupstream from the poly(A) sequence at the 3′ end of the cDNA.

cDNA expression plasmids were constructed using standard cloning methodswell known in the art, by introducing the T11-related cDNA encompassingnucleotides 1 to 3454 (FIG. 3) into the pSV2 gpt vector into which thesimian sarcoma virus long-terminal—repeat (LTR) had been engineered asthe promoter, as previously described in detail (49).

DNAs and sense strand RNAs of this invention can be employed, inconjunction with protein production methods known in the art, to producecells expressing functional type α PDGF-R protein from the novel gene inthe absence of other PDGF receptors. These novel receptors can be usedfor functional studies in cells, such as qualitative and quantitativereceptor binding assays.

Accordingly, one embodiment of this aspect of this invention comprises acell, preferably a mammalian cell, transformed with a DNA of theinvention, wherein the transforming DNA is capable of being expressed.Mammalian cells (COS-1) transformed with the pSV2 gpt vector carrying aT11-related cDNA were prepared according to well-known methods and wereshown to express T11 gene products as 185 kd and 160 kd species (FIG.7B). These products were capable of binding human PDGF isolated fromplatelet, as illustrated in the Experimental Section below (see, FIG.8).

Additional work in the Experimental Section demonstrates further thatDNAs of this invention can be used to reconstitute type α PDGF receptorgene function in other cells free of PDGF receptors, and that eachreceptor type, α or β, efficiently mediates major known PDGF activitiesincluding mitogenic signal transduction, chemotaxis and stimulation ofphosphoinositide turnover. Moreover, these studies further establish thetype α PDGF receptor as the principal receptor for the main form ofhuman PDGF which is derived from platelets.

Thus, by so using the DNAs of the invention in gene expression methods,especially the preferred TR4 cDNA clone listed herein, those skilled inthe art, without undue experimentation, can construct cell systems whichfall within the scope of this invention, for determining the mechanismsof PDGF regulatory processes, as well as for production of large amountsof the novel PDGF receptor protein.

This invention further comprises novel bioassay methods for detectingthe expression of genes related to DNAs of the invention. According toone such embodiment, DNAs of this invention may be used as probes todetermine levels of related mRNAs. This embodiment is exemplified by thecomparison of mRNA species of the T11 and known PDGF-R genes in normaland tumor cells (FIG. 6). Total or polyadenylated RNA was separated bydenaturing gel electrophoresis in formaldehyde (48), transferred tonitrocellulose, and hybridized under stringent conditions with³²P-labeled probes. The probes were prepared from any of the followingDNAs of this invention: clone pT11-HP (0.95-kbp HindIII-PstI fragment ofgenomic clone T11) or from T11 cDNA (3.5-kbp BamHI fragment of TR4,including the whole coding region).

Therefore, by employing the DNAs and RNAs of the invention in knownhybridization methods, especially the most preferred DNAs listed herein,those skilled in the art, without undue experimentation, can measurelevels of expression of type α PDGF-R gene without interference frommRNA of type β PDGF-R gene or other related oncogenes.

This invention also comprises novel antibodies made against a peptideencoded by a DNA segment of the invention or by other related DNAs. Thisembodiment of the invention is exemplified by rabbit antisera containingantibodies which specifically bind to type α PDGF-R protein or, in thealternative, to the known PDGF-R protein, herein designated type β.

Such type specific antisera were raised to synthetic peptidesrepresenting 15 amino acid sequences from the carboxyl-terminal regionsof their respective PDGF-R proteins (residues 959-973 of the type αsequence displayed in FIG. 3, and corresponding residues 967-981 of theknown type β sequence, as predicted by the respective cDNA sequences).These peptides were selected to meet the following criteria: lack ofsequence relatedness between the two PDGF-R types (less than 50%sequence homology); relative hydrophilicity; and carboxyl -terminallocation which is known to be associated with a higher likelihood ofproducing antibodies reactive with native proteins.

Antisera to peptides were prepared by chemically synthesizing thepeptides, conjugating them to carrier (thyroglobulin), and injecting theconjugated peptides into rabbits with complete Freund's adjuvant,according to standard methods of peptide immunization.

These antibodies can be used for detection or purification of theprotein products. Thus, FIG. 7 shows the use in Western blot experimentsof two different rabbit antibodies (anti-T11 (PDGF-R type α) andanti-HPR (PDGF-R type β)] raised against the corresponding type-specificpeptides. As is evident from th figure, the appropriate PDGF-R types arespecifically detected in various cells by antisera from rabbitsimmunized with synthetic peptides.

Experimental Section

This section describes experimental work leading to the identificationand cloning of a genomic sequence and cDNAs of a novel receptor-likegene of the PDGF receptor/CSF-1 receptor subfamily. The gene gives riseto a 6.4-kb RNA transcript that is coexpressed in normal human tissueswith the known 5.3-kb PDGF receptor mRNA. The new PDGF receptor gene waslocalized to chromosome 4 at location 4q 11-12, consistent with theclustering of other genes of this receptor subfamily on ancestrallyrelated chromosomes 4 and 5.

That the cloned cDNA is functional is demonstrated by the observationthat introduction (by transfection using a viral vector) of a cDNA ofthe novel gene into COS-1 cells leads to expression of proteins whichare specifically detected with anti-serum directed against a predictedpeptide. Transfected but not control COS-1 cells demonstrate specificbinding of ¹²⁵I-human PDGF, which is efficiently competed by all threePDGF isoforms, including the main AB form found in human platelets. Incontrast, expression of the known PDGF receptor cDNA in COS-1 cellsleads to PDGF binding with a distinct pattern of competition by the samePDGF isoforms characterized by a marked preference for PDGF form BB.

Further evidence that the new receptor gene encodes a distinct PDGFreceptor derives from examination of human cells, originally free ofPDGF receptors, in which PDGF-receptor activities are reconstituted byeither type α or type β receptors introduced by transfection withvectors bearing the respective cDNAs. Cells with the type α receptorsare significantly more responsive to PDGF-AB in all of the followingPDGF-mediated cellular activities: tyrosine phosphorylation of thereceptor gene product; stimulation of DNA synthesis and consequent cellproliferation; chemotaxis; phosphoinositide breakdown; and cytosoliccalcium mobilization ([Ca²+]i).

Thus, while each type of reconstituted PDGF-R gene product independentlyelicits similar biochemical as well as biological responses to PDGF-BB,the type α PDGF-R is the preferred receptor for PDGF-AB, the principalisoform of human PDGF which is found in platelets. Accordingly, itfollows that abnormalities in the structure or expression of the type αPDGF receptor could have profound pathological effects for which thepresent invention provides means of diagnosis and therapy.

Materials and Methods

Detection of v-fms and 2DGF receptor-related gene fragments in-humanplacenta and thymus DNAs. Genomic DNA (20 μg) was digested with EcoRI,separated by electrophoresis in 0.8% agarose gels, and transferred tonitrocellulose paper (41). Hybridization to ³²P-labeled probes (42) wasconducted in a solution of 50% or 30% formamide, 0.75 M NaCl, and 0.075M sodium citrate, at 42° C. (43). After hybridization, the blots werewashed in 2×SSC (0.3 M NaCl; 0.03 M sodium citrate) at room temperature,and then in 0.1× or 0.6×SSC at 50° C. (stringent or relaxed condition,respectively). The v-fms probe was a 0.44-kbp XhoI-BglI fragmentencompassing nucleotides 3891 to 4419 of the v-fms oncogene (44). Themouse PDGF receptor probe was a 0.5-kbp SinI-PvuI fragment encompassingnucleotide 2490 to 2995 of its cDNA (6).

Molecular cloning of the 8T11 genomic fragment as well as cDNAs of T11and PDGF-R genes. Libraries from which specific cDNA clones (inparentheses) were isolated included: human fibroblast mRNAs in theOkayama-Berg vector (pHF); human infant brain mRNAs in λgt11 (λαHB)human embryonic fibroblast random primed mRNAs in λgt11 (λEF); and humanembryonic fibroblast mkNAs in the directional cloning phagemid (TR4 orHPR) Restriction sites were determined by electrophoretic analysis ofthe products of single and double digestions. Regions of λT11 homologousto the v-fms or mouse PDGF receptor probes were identified byhybridization as described in FIG. 1. Three restriction fragments(0.95-kbp HindIII-PstI 0.5-kbp AvaI-SacI, and 0.35-kbp KpnI-XbaI)including regions homologous to the v-fms and mouse PDGF receptor probeswere subcloned into plasmids and sequenced by the dideoxy chaintermination method (45).

Chromosome mapping of the T11 gene. The probe was labeled with all four³H-nucleotides (New England Nuclear, Boston, Mass.) using a modifiednick translation kit (Amersham, Arlington Heights, Ill.) to a specificactivity of 2.5×10⁷ cpm/μg DNA. In situ hybridization with humanmetaphases and prometaphases from methotrexate-synchronized peripherallymphocyte cultures was carried out as previously described (47).

Comparison of mRNA-species by Northern blot hybridization. Total orpolyadenylated RNA was separated by denaturing gel electrophoresis informaldehyde (48), transferred to nitrocellulose, and hybridized understringent conditions (50% formamide, 0.075M NaCl, 0.75M sodium citrate,at 42° C.) with ³²P-labeled probes.

Detection of T11 and PDGF-R proteins with peptide antisera. Anti-T11 andanti-PDGF-R sera were obtained following immunization of rabbits with 15amino acid peptides from the corresponding carboxyl-terminal regions ofthe predicted receptors. These peptide sequences were less than 50%homologous. cDNA expression plasmids were constructed by introducing theT11 cDNA encompassing nucleotides 1 to 3454 (FIG. 3) or the PDGF-R cDNAencompassing nucleotides I to 3939 into the pSV2 gpt vector into whichthe simian sarcoma virus LTR had been engineered as the promoter (49).About 10⁶ COS-1 cells in 10 cm petri dishes were incubated in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal calf serum 24hr prior to transfection. DNA transfection was performed by the calciumphosphate precipitation method (50) 48 hours prior to analysis. Cultureswere lysed with staph-A buffer (10 mM sodium phosphate pH7.5, 100 mMNaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 0.1% aprotinin, 1 mMPMSF, and 1 mM sodium orthovanadate) and clarified by centrifugation at10,000×g for 30 min. Proteins (100 φg per lane) were resolved byelectrophoresis in 7% SDS-polyacrylamide gels, transferred tonitrocellulose filters and probed by immunoblot analysis (with orwithout peptide blocking) using ¹²⁵I-Protein A (51).

Binding of ¹²⁵I-labeled human PDGF to receptors on cells. COS-1 cellswere plated in 12-well plates and transfected 48 hours before assay asdescribed in FIG. 7. Human PDGF was labeled with ¹²⁵I by thechloramine-T method to specific activities of 3.7×10⁴ cpm/ng (52). Thebinding of ¹²⁵I-labeled PDGF isolated from human platelets (53) in theabsence or presence of a 50-100 fold excess of unlabeled human PDGF (AB)(Collaborative Research), recombinant PDGF-BB (AmGen) or recombinantPDGF-AA (37), was carried out at 4° C. for 2 hrs. Unbound ¹²⁵I-PDGF wasremoved by four successive washes with binding buffer (DMEM containing 1mg per ml bovine serum albumin). The cells were then lysed insolubilizing buffer (1% Triton X-100, 20 mM Hepes pH 7.4, 10% [v/v]glycerol), and radioactivity measured with a T counter.

Tyrosine autophosphorylation of type α and type β-PDGF-R gene products.After incubation with PDGF for 5 min at 37° C., cell lysates wereimmunoprecipitated with anti-peptide antisera. Total cell lysates orimmunoprecipitates were analyzed by immunoblotting with antibodies tothe receptors or to phosphotyrosine (anti-P-Tyr) (54). Theanti-phosphotyrosine antibodies were preincubated with 10 mMphosphotyrosine for blocking.

Results

Detection of a novel human PDGF-R/CSF1-R-related gene. In order toexplore novel sequences related to known growth factor receptor genes ofthe PDGF-R/CSF1-R family, high molecular weight DNAs prepared from humanplacenta and thymus were digested with EcoRI and analyzed by blothybridization with DNA probes derived from the tyrosine-kinase domainsof v-fms and the mouse PDGF-R gene (FIG. 1). Under stringent conditions,the v-fms probe detected EcoRI restriction fragments of 27-kbp and/or20-kbp, due to the previously reported restriction polymorphism at thislocus (8). Under less stringent conditions, several additional fragmentsof 12-, 6.8-, 5-, 2.7-, 2.2-kbp, which hybridized to the v-fms probe,were observed. The corresponding region of the mouse PDGF-R cDNAhybridized with a single 21-kbp fragment under stringent conditions(FIG. 1).

At lower stringency, the same probe detected several additionalfragments, some of which had sizes similar to those of the v-fms-relatedfragments described above. Among these, the 12-kbp EcoRI fragmenthybridized more strongly than the other fragments with both probes.Moreover, some of the smaller bands corresponded to restrictionfragments reported for human c-kit (7). Thus, it was decided to clonethe 12-kbp EcoRI DNA fragment and characterize it more fully.

Using the XEMBL-4 vector system, a genomic library size-selected bysucrose gradients was constructed from EcoRI-digested normal humanthymus DNA. FIG. 2 shows the restriction map of λT21 containing a 12-kbpEcoRI insert, which hybridized with both v-fms and mouse PDGF-R probesonly under relaxed but not stringent hybridization conditions. Regionshomologous to v-fms/PDGF-R tyrosine kinase domains were localized byhybridization to restriction endonuclease digests of λT11 DNA.

Three plasmid subclones containing sequences hybridizing to the 0.95-kbpHindIII-PstI 0.5-kbp Aval-SacI, and 0.35-kbp KpnI-XbaI fragments of λT11were subjected to nucleotide sequence analysis. Their discrete openreading frames (FIG. 3) showed relatedness to both human c-fins andmouse PDGF-R genes, but were readily distinguished from each of thesegenes (3,6) as well as from c-kit (7). The three putative coding regionswere each flanked by the AG and GT dinucleotides that border the exonsof eukaryotic genes (9).

To assess whether the T11 sequence was transcribed, Northern blotanalyses of a variety of cells were performed using a clone of the0.95-kbp HindIII-PstI fragment (pT11-HP) which contained exon (a) (FIG.2) and lacked human repetitive sequences. Under stringent conditions, asingle 6.4-kb RNA transcript was detected in poly(A)+ RNA prepared fromnormal human fibroblasts (data not shown). This transcript differed insize from previously reported transcripts for the PDGF-R (6), c-fms (3)or c-kit genes (7). All of these findings indicated that the T11sequence represented a gene distinct from known members of thissubfamily of tyrosine kinase receptors.

cDNA cloning of the novel gene. A normal human fibroblast cDNA libraryin the Okayama-Berg expression vector was initially screened understringent conditions using the pT11-HP clone of the 0.9-kbp HindIII-PstIfragment of λT11. One strongly hybridizing clone containing a 3.9-kbpcDNA insert was isolated (FIG. 2). This clone, designated pHF1,hybridized to a 6.4-kb transcript in normal human fibroblasts andcontained a polyadenylation signal followed by a poly(A) tail at its 3′end. It also contained the coding sequence within the λT11 DNA and 170nucleotides related to CSF1-R-PDGF-R tyrosine kinase domains upstream ofexon (a).

The 0.4-kbp 5′ end of pHF1 was used to search for overlapping cDNAclones in a human infant brain library. Under stringent conditions, anumber of positive clones with similar restriction maps were isolated(data not shown). The longest, λHB6, (FIG. 2) was subjected to sequenceanalysis. A possible ATG initiation codon was identified within anotherclone, kEF17, isolated by screening a M426 human embryo fibroblast cDNAlibrary in the λgt11 vector with a 0.2-kbp 5′ fragment of λHB6 as aprobe. The three overlapping clones (pHF1, λHB6 and λEF17) contained theentire coding region in addition to 138-bp 5′ and ˜3-kbp of 3′untranslated sequences (FIG. 2).

Two clones, λHB3 and λHB4, that gave weaker signals in plaquehybridization during screening of the human infant brain library werealso sequenced. These showed close similarity to the sequence of themouse PDGF-R cDNA (6). Moreover, when the 2.0-kbp insert of HB4 washybridized to normal human fibroblast RNA, it detected a transcript of5.3-kb, consistent with that of the PDGF-R (6).

No clones containing sequences further upstream from the 5′ end of λHB4could be obtained by screening the human infant brain cDNA library inλgt11. This was accomplished by utilizing a M426 human embryo fibroblastcDNA library in a new phagemid vector constructed as described elsewhere(10). By screening this library with a 0.3-kbp 5′ subfragment of λHB3,two overlapping clones, HPR2 and HPR5, were obtained. These containedbetween them the entire known human PDGF-R coding sequence, its complete3′ untranslated region, and 360 nucleotides of its 5′ untranslatedregion (FIG. 2). A 6.4-kbp cDNA clone (TR4) of the novel related genewas also obtained from this same library by screening with a 5′ 0.2-kbpsubfragment of λEF17.

Deduced amino acid sequence establishes the T11 gene as a member of thePDGF-R/CSF1-R subfamily. The complete nucleotide sequence of the 6.4-kbpcDNA of the T11 gene is shown in FIG. 3. An open reading frame beginningwith a possible ATG initiation codon at nucleotide position 139 extendedto a TAA termination codon at position 3406. Although the open readingframe extended further upstream, the putative initiation ATG was flankedby sequences that fulfill the Kozak criteria for an authentic initiationcodon (11). Moreover, the first 23 amino acid stretch displayedproperties of a cleavable hydrophobic signal peptide (FIGS. 3 & 4). Atthe 3′ end, the open reading frame was followed by ˜3-kbp ofuntranslated sequences. A polyadenylation signal (AATAAA) was located 25nucleotides upstream from the poly(A) sequence at the 3′ end of theCDNA.

According to the putative cleavage site for the signal peptide (12), theamino terminus of the mature product was predicted to be glutamine atamino acid 24 followed by 1066 amino acids. This polypeptide sequencewith a calculated molecular mass of around 120 kd contained all of thecharacteristics of a membrane-spanning tyrosine kinase receptor. Ahydrophobic segment consisting of 24 amino acids (residues 525 to 548)exhibited characteristics of a receptor transmembrane domain (FIGS. 3 &4). Between the signal peptide and the transmembrane domain, there wasstructural homology with the extracellular ligand binding domains of thePDGF-R/CSF1-R subfamily. Ten cysteine residues were spaced at the samepositions as in the other receptors of this subfamily, and eightpotential N-linked glycosylation sites were distributed in its putativeextracellular domain (FIG. 3).

The cytoplasmic domain was comprised of a conserved tyrosine kinaseregion and a hydrophilic carboxyl-terminal tail (FIGS. 3 & 4). Thetyrosine kinase domain included the consensus ATP binding sequence(residues Gly-X-Gly-X-X-Gly . . . Lys) and a tyrosine residue atposition 849 homologous to the major autophosphorylation site ofpp60^(V-src) at position 416 (13). Moreover, the tyrosine kinase wasdivided into two domains by a hydrophilic inter-kinase sequence aspreviously shown for c-fms/CSF1-R, PDGF-R, and c-kit (FIG. 4).

The amino acid homologies of its extracellular domain with those of thePDGF-R, CSF1-R, and c-kit were 31%, 18%, and 19% respectively. The twokinase domains of the T11 gene were most homologous to those of thehuman PDGF receptor (85% and 75%, respectively) as compared with 67 to70% for c-fms and c-kit (FIG. 4). Even in the inter-kinase domain, itsamino acid sequence was more closely aligned to the PDGF-R with 27%homology compared to 10 and 19% with c-fms or c-kit. These observationslead to the conclusion that the T11 product was in the PDGF-R/CSF1-Rsubfamily and most closely related to the PDGF-R.

The deduced amino acid sequence of another cDNA clone (obtained in thesame experiment which produced the TR4 cDNA clone) established itsproduct as the known human PDGF receptor. Its sequence correspondedalmost completely with the recently published sequence of the knownhuman PDGF receptor (14). A single nucleotide difference changed residue240 from Asn to Ser. Comparison with the mouse PDGF receptor cDNA aminoacid sequence also revealed high similarities throughout all functionaldomains including the ligand binding domain (79%), transmembrane domain(96%), the juxtamembrane domain (97%), split tyrosine kinase domains(TK1, 99% and TK2, 97%), inter-kinase domain (86%) and carboxyl terminus(85%).

Chromosomal mapping of the T11 gene. To define the new gene with respectto chromosomal location, 104 chromosome spreads were examined by in situhybridization with a pT11-P probe. A total of 136 grains were localizedon a 400-band ideogram (FIG. 5). Of the total grains, 50 (37%) were onchromosome 4 with the majority of 45 grains tightly clustered near theacentromeric region of the long arm at bands, q11-12 (FIG. 5). A secondsite of hybridization on chromosome 5q 11.1-11.2 consisting of 7 grainsaccounted for 5% of the total grains (FIG. 5).

The T11 gene probe was also hybridized to chromosomes derived from aBurkitt lymphoma cell line carrying a large abnormal marker chromosomeoriginating from a translocation t1;5 (p22; q23) translocation. Therewas no detectable labeling of the rearranged chromosome 5 in over 300spreads examined for the presence of grains at this chromosome. Thus, insitu hybridization assigned the T11 gene to chromosome 4 at locationq11-12. This localization places the new gene within the same region asthe c-kit proto-oncogene (15). The structurally related genes forplatelet factor 4, (16), interferon τ-inducible factor; τIP-10, (17) andmelanoma growth stimulatory activity (MGSA) (18) as well as genes forα-feto protein, albumin (19), HPAFP (20), and the gene fordentinogenesis imperfecta have been mapped at 4q 11-13 (21).

Expression of transcripts and protein products of the endogenous T11gene in normal and tumor cells. To investigate the tissue specificexpression of the new receptor-like gene, either of the most preferredDNAs of this invention, i.e., the HindIII-PstI. 0.95-kbp fragment of theT11 genomic clone, or cDNA insert of TR4, was used for Northern blothybridization experiments. A single 6.4-kb transcript was detected inpoly(A)-containing RNAs of a variety of human tissues and cell lines. Asshown in FIG. 6, relatively high levels of the transcript were found insmooth muscle, heart, and human embryo, while human liver and spleendemonstrated undetectable or barely detectable transcripts under theseconditions.

Using a probe for the known human PDGF receptor gene, it was noted thatthe T11 and 5.3-kb PDGF-R transcripts appeared to be coexpressed atsimilar respective levels in each of these same tissues. Human skeletalmuscle, fetal brain, placenta as well as cultured fibroblasts and glialcells also expressed high levels of both transcripts (data not shown).

Thus, the new gene and the known PDGF-R gene appeared to be coordinatelyexpressed in normal tissues examined and exhibited a very differentpattern from that reported for either c-fms/CSF1-R or c-kit (3,7).

Expression of the T11 and PDGF-R genes were also compared in human tumorcells. Here, their patterns of expression could be readilydistinguished. Several tumor cell lines were found to contain one or theother transcript but not both (FIGS. 6C and D).

Antibodies specific for either the novel or known PDGF receptor protein.In an effort to identify the protein product of the new gene, antiserato peptides were prepared based on its predicted sequence. Analogousregions of the predicted sequence of the known PDGF-R were utilized togenerate antisera as well. Initial efforts to detect specific expressionof the T11 gene product utilized M426 embryo fibroblast cells, fromwhich cDNAs of both receptors had been isolated. 8387 and A204 celllines which specifically expressed the PDGF-R or T11 gene transcripts,respectively were analyzed as well (FIG. 7A).

Western blot analysis of M426 cells with antisera (anti-T11) directedagainst the T11 gene product revealed 180 kd and 160 kd protein species,which were specifically competed by the immunizingpeptide. Theanti-PDGF-R peptide serum (designated anti-HPR) detected 180 and 165 kdproteins in the same cells. Western blot analysis of 8387 cells revealed180 and 165 kd species, which were recognized by the anti-HPR, but notby anti-T11 serum. Conversely, A204 cells contained 180 and 160 kdspecies which were specifically detected by anti-T11, but not recognizedby anti-HPR serum.

All of these findings indicated that these antibodies of this inventionwere specific for detection of the homologous receptor gene product andthat T11 gene products were expressed in cells containing itstranscript.

Expression of T11 cDNA in a mammalian vector system. As further test ofthe ability to immunologically detect the T11 gene product as well as toinvestigate the functional expression of its cDNA, LTR-based expressionvectors were constructed for the T11 cDNA encompassing nucleotides 1 to3454 (FIG. 3) and for the corresponding known PDGF-R cDNA as well.

Transient expression in COS-1 cells led to the specific detection of theT11 gene products as 185 kd and 160 kd species (FIG. 7B) whereas thePDGF-R appeared as 185 kd and 165 kd proteins. The respective lower MWforms of each receptor did not vary in size among the cells analyzed.However, some different sizes of the higher MW species were observed,which were likely due to cell specific differences in glycosylation.

PDGF binding to the T11 product establishes it as a new PDGF-R gene.Because of their structural and deduced amino acid sequence similaritiesas well as their coexpression by normal cell types known to respond toPDGF, to studies were performed to determine whether the T11 geneproduct exhibited any functional relationship to the known PDGF-R geneproduct. Thus, ¹²⁵I-labeled human PDGF was incubated with control andtransfected COS-1 cells in the presence or absence of unlabeled PDGFisoforms.

As shown in FIG. 8, as much ¹²⁵I-PDGF specifically bound to COS-1 cellstransfected with the new receptor gene as to NIH/3T3 cells. Binding wasreduced to the level of nontransfected COS-1 cells by competition withexcess human PDGF (predominantly AB), PDGF-BB, or PDGF-AA. Specificbinding of ¹²⁵I-PDGF to COS-1 cells transfected with the PDGF-R cDNA wasalso observed. In this case, however, binding was competed by human PDGF(i.e., PDGF-AB) and PDGF-BB but not by PDGF-AA (FIG. 8).

Thus, while both T11 gene and PDGF-R gene products bound human PDGF, thepattern of competition by different PDGF isoforms distinguished the tworeceptors. These results implied that the T11 gene encoded a novel PDGFreceptor with different affinities for the three dimeric forms of PDGF.Hence, the T11 receptor gene product was tentatively designated as thetype α because PDGF binding was competed by AA as well as BB isoforms,and the product of the previously cloned PDGF receptor was designated astype β.

PDGF isoforms induce different patterns of autophosphorylation of thenovel and known PDGF receptors. After PDGF binding to its receptor, anumber of molecular events are rapidly triggered in vivo, includingphosphorylation of the receptor protein on tyrosine residues (22). Tocompare the relative autophosphorylation of the products of the twoPDGF-R genes by each PDGF isoform, the responses of A204 and 8387 cellsthat expressed type α and type β PDGF-R genes, respectively, wereanalyzed.

As shown in FIG. 9A, immunoblots of A204 cells lysed 5 minutes followingligand exposure revealed readily detectable and very similar levels ofautophosphorylation of a 180 kd species in response to each of the threePDGF isoforms. As further evidence that the induced autophosphorylationwas specific to the type α receptor gene product, ligand stimulated A204cell lysates were first subjected to immunoprecipitation with anti-typeα PDGF-R serum (anti-T11) followed by immunoblotting withanti-phosphotyrosine serum. By this approach, it was firmly establishedthat the 180 kd type α PDGF receptor was phosphorylated on its tyrosinewith similar intensity in response to each of the three ligands.

Exposure of 8387 cells, which expressed only the type α PDGF geneproduct, to the same amount of each respective PDGF isoform revealed avery different pattern of receptor autophosphorylation. Here, PDGF-BBinduced the highest level of autophosphorylation of the 180 kd speciesspecifically recognized by anti-type β PDGF-R serum (anti-HPR), andhuman PDGF induced detectable autophosphorylation as well (FIG. 9B). Incontrast, PDGF-AA induced no detectable phosphorylation.

Thus, while PDGF-AB and PDGF-BB triggered both receptors, the muchstronger response of the β type receptor to the BB homodimer as well asits lack of detectable response to the AA homodimer readilydistinguished the receptors functionally.

To investigate the pattern of autophosphorylation of the two receptorsby different PDGF isoforms in the same cells, NIH/3T3 cells were firsttriggered by different ligands followed by immunoprecipitation witheither anti-type α or β PDGF-R serum. The immunoprecipitated receptorproteins were then analyzed by immunoblotting with anti-phosphotyrosineserum.

As shown in FIG. 9C, the 180 kd protein immunoprecipitated by the type αPDGF-R antiserum was phosphorylated by all three dimeric forms of PDGF.In contrast, the 180 kd phosphoprotein immunoprecipitated by theanti-type β receptor serum was detected only after human PDGF-AB orPDGF-BB stimulation. Thus, the patterns of response to different PDGFligands, remained receptor-specific in at one example of nontransformedcells naturally expressing both PDGF-R genes.

Type a PDGF receptor is more-efficient in stimulating DNA synthesis inresponse to PDGF isoform AB. The expression of the two receptors inother fibroblast lines was analyzed next. Western blotting analysis(data not shown) revealed significant variations in the ratio of the tworeceptors among the lines analyzed. Whereas mouse fibroblasts expressedsimilar levels of type α and type β receptors, human fibroblasts such asAG1523 or M413 expressed relatively lower levels of the type α receptorthan either mouse fibroblasts or M426 human fibroblasts.

Saturating amounts of PDGF-AB or PDGF-BB yielded similar increases inDNA synthesis in each of the cell lines (data not shown). However,submaximal doses of PDGF-AB and PDGF-BB showed significant differencesin the levels of mitogenic activity observed (FIG. 10). Whereas,NIH/3T3, BALB/3T3 and M426 cells responded with comparable efficiency toPDGF-BB and AB, PDGF-AB was significantly less active on AG1523 or M413cells. Their lesser mitogenic responsiveness to PDGF-AB seemed tocorrelate with the high ratio of β to α receptors in these cellsdetected immunologically.

Taken together with the dose-response curves observed forphosphorylation of the two receptors in NIH/3T3 cells by the differentPDGF isoforms, these results strongly suggested preferential triggeringof the type α receptor, in the presence of the type β receptor, byPDGF-AB, as well as by PDGF-AA.

Independent expression of two PDGF gene types after introduction ofcDNAs into PDGF receptor-free hematopoietic cells. To investigate thebiological and biochemical responses specific to each PDGF-R geneproduct, systems were developed to look at this receptor in cells inwhich each type could be independently introduced and expressed. Thesesystems were based on the 32D cell line, a mouse hematopoietic cell linenormally dependent on I1-3 for survival and proliferation. Recentstudies have established that introduction of an expression vector forthe EGF-R in these cells led to effective coupling with EGF mitogenicsignal transduction pathways.

The mammalian expression vectors described above, carrying the gptselectable marker, was used to transfect 32D cells with either the typeα or the type β PDGF-R cDNAs by electroporation. Transformants wereselected using medium supplemented with mycophenolic acid. After 2 weeksin the selective medium, viable cultures were obtained.

Cultures designated 32D-αR and 32D-βR, respectively were subjected toNorthern blot analysis, as described above. Neither type of PDGF-R mRNAwas detectable in the parental 32D cells even under relaxedhybridization conditions, which conditions enabled detection of therespective mouse PDGF-R gene transcripts in NIH/3T3 fibroblasts. Incontrast, 32-αR and 32D-βR transfectants expressed abundant transcriptsspecific to the human type α and type β PDGF-R genes, respectively. Whenmembrane lysates of these transfectant were subjected to immunoblotanalysis, anti-type α PDGF-R peptide serum detected 180 kd and 160 kdprotein species in 32D-αR but not in 32D-β cells. Moreover, theseproteins were specifically competed by the immunizing peptide.Conversely, 32D-βR cells contained 180-200 kd and a 165 kd species whichwere specifically detected by the anti-type β PDGF-R serum. None ofthese proteins species were detectable in control 32D cells.

Type α receptor has a higher binding affinity for the PDGF-AB isoform.PDGF-BB binding was compared in 32D-αR or 32D-βR transfectants, and bothshowed high affinity binding. Scatchard analysis revealed about twothousand receptors per cell with a single affinity class of bindingsites. The Kds were 0.4 nM and 0.5 nM for 32D-αR and 32D-βR cells,respectively (FIG. 11). 32D-αR cells also showed a high binding affinity(K_(d)=0.4 nM) for ¹²⁵I-PDGF-AB, exhibiting the same number of bindingsites as for PDGF-BB.

In contrast, however, 32D-βR cells revealed ten times less bindingcapacity for ¹²⁵I-PDGF-AB than did 32D-αR cells. Thus, standardized onthe basis of their similar binding of PDGF-BB, the type β receptorshowed a strikingly lower affinity for PDGF-AB.

Common biological functions independently triggered by type α and β PDGFgene products. Mitogenesis and chemotaxis are among the most wellcharacterized responses of fibroblasts to PDGF. Thus, whether 32D-αR orβR lines mediated either of these biological responses was investigated.

Growth of 32D cells is normally strictly dependent on IL-3, anddeprivation of IL-3 from the medium led to the rapid loss of viabilityboth of the transfectants and the control 32D cells. As shown in FIG.12, PDGF-BB was able to couple efficiently with mitogenic signaltransduction pathways and abrogate IL-3 dependence in a similar doesdependent manner in both transfectants, but had no effect in control 32Dcells. Thus, the presence of either type α or β. PDGF-R was bothnecessary and sufficient for the mitogenic response to PDGF BB.

However, whereas, the type α receptor containing 32D cells were asresponsive to PDGF-AB as to PDGF-BB, PDGF-AB elicited a significantlylesser DNA synthesis response in 32D-βR cells (FIG. 12).

These findings were confirmed by analysis of colony-formation insemi-solid agar containing medium. Both transfectants formed coloniesreadily in PDGF-BB, supplemented medium but only 32D-∀R cells did so inmedium supplemented with PDGF-AB (data not shown). Thus, the mitogenicresponses observed with both 32D-αR and βR transfectants correlated wellwith the binding properties of the same PDGF isoforms to α and βreceptors expressed by each cell line, respectively.

To address whether chemotaxis was specifically mediated by either type αor PDGF receptors, a chemotaxis assay was employed using the modifiedBoyden chamber technique well known in the art. While 32D cells lackingPDGF receptors did not respond to PDGF-AB or PDGF-BB, PDGF-BB waschemotaxic for both α and β receptor expressing transfectants. PDGF-ABwas relatively more active on 32D-αR cells (FIG. 13).

Thus, each PDGF receptor independently coupled with both mitogenic andchemotaxis signalling pathways inherently present in 32D cells.Moreover, these functions were triggered according to the relativebinding abilities of PDGF isoforms to either receptor.

Inositol lipid metabolism and cytosolic Ca²⁺ mobilization coupling withindependently reconstituted receptors. Recent investigations havesuggested an important role of receptor-mediated turnover of inositollipids resulting in the increase of second messengers such asintracellular free calcium and diacyloglycerol in the transduction ofthe PDGF-induced mitogenic signal. Thus, the effects of PDGF-AB andPDGF-BB on inositol lipid metabolism and intracellular free Ca²⁺([Ca²⁺]i) were studied in type α and type β PDGF-R containing 32D cells.

The accumulation of radioactive inositol phosphates was measured afterprelabelling cultures with ³H-myoinositol and challenge with PDGFisoforms at 37° C. in the presence of LiCl, according to methods wellknown in the art. [Ca²⁺]i was measured in 32D cells in suspension,loaded with the fluorescent [Ca²⁺]i indicator fura-2, and treated withPDGFs in the complete incubation medium.

FIG. 14 shows the effect of PDGF-AB and PDGF-BB on inositol phosphateformation and [Ca²⁺]i in type α and type β PDGF-R 32D cells. As shown inFIG. 14 (panel A), both PDGF-BB and PDGF-AB were able to elicitdose-dependent accumulation of inositol phosphates, with similarrelative potencies. The same isoforms exerted almost identical increasesin [Ca²⁺]i in type α PDGF-R 32D cells as well (FIG. 14, panel A,insert). PDGF-BB also markedly stimulated inositol lipid metabolism andintracellular Ca²⁺ mobilization in type β PDGF-R 32D cells, establishingthe very similar biochemical responses elicited by these distinct PDGF-Rgene products in 32D cells in response to PDGF-BB.

FIG. 14 (panel B) shows that PDGF-AB was significantly less effectivethan PDGF-BB in promoting inositol phosphate accumulation in type βPDGF-R 32D cells. Detectable release of inositol phosphate occurred onlyat high PDGF-AB concentration. Similarly, PDGF-AB elicited little or no(Ca²⁺)i response.

Discussion

The present studies demonstrate the existence of two distinct human PDGFreceptor genes. Further, they illustrate the detection and isolation oftwo principal embodiments of this invention, the genomic and cDNA clonesof a novel gene within the PDGF-R/CSF1-R subfamily. This gene isdivergent from but most closely related to the known PDGF-R gene. Underconditions of natural expression as well as following introduction ofthis novel cDNA into appropriate target cells by means of an expressionvector, functional responses of its product to PDGF were demonstrated atconcentrations that bound and triggered tyrosine phosphorylation of thepreviously identified PDGF receptor.

Standardized on the basis of similar levels of tyrosine phosphorylation(and several other activities) of PDGF-R gene product induced by aconstant amount of PDGF, the new receptor was shown to respond betterthan the known PDGF-R to the AA homodimer. Conversely, the knownreceptor responded preferentially to the BB homodimer. Based upon thepresent findings, the new gene product has been designated as the type αPDGF-R and the previously identified PDGF-R gene product as the type βreceptor.

The AA homodimer failed to stimulate detectable tyrosine phosphorylationof the β type receptor in NIH/3T3 cells and yet is capable of inducingDNA synthesis in this cell line (37). This indicated that the α typereceptor can couple with mitogenic signalling pathways in fibroblasts.The β type receptor has also been reported to couple PDGF with mitogenicpathways (28). These results suggested that both receptor gene productscan induce a proliferative response.

The ability, according to compositions and methods of this invention, tostably introduce expression vectors for these distinct receptor genesinto a null cell made it possible to confirm this suggestion in humancells. Further studies in such cells showed that other known PDGFfunctions including chemotaxis (38), membrane ruffling (39), as well astransmodulation of a heterologous receptor (40), are not specificallymediated by either type α or β PDGF-R gene products.

Such knowledge is a necessary prelude to understanding and diagnosis ofdisease conditions affecting these PDGF functions, which can befurthered through additional practice of the present invention.

Among human tumor cell lines analyzed using methods of this invention,several were observed in which there was discoordinate expression of thetwo PDGF-R genes. Moreover, representative tumor cell lines expressingmRNA from either gene were shown to contain the respective proteinproduct, which bound and was phosphorylated on tyrosine in response toPDGF.

The availability of the immunologic as well as the molecular probes ofthis invention, specific for either type α or type β PDGF-R geneproducts, makes it possible to identify human tumors in which expressionof the PDGF-A or B chain, in combination with either receptor gene, maybe causally implicated in tumor development. At the same time, theavailability of reagents for specific detection of each type ofcomponent is a critical aid in efforts to implicate the abnormalexpression of this complex growth factor-receptor network in otherchronic disease states such as arteriosclerosis, arthritis, and fibroticdiseases (23).

Additional observations of scientific import have already been providedby the practice of the invention as herein described. For instance, thechromosomal location of the novel gene, established using DNAs of thisinvention, provides insight into the possible evolution of this receptorgene family. Thus, the chromosomal localization places the type α PDGFreceptor gene on chromosome 4 at 4q 11-12, the same region as c-kit(15), a related receptor-like gene. Other genes of this subfamily havebeen localized on chromosome 5. These include the type β PDGF-R mappedat 5q 23-31 (6) and the CSFI-R gene, on 5q 33.2-33.3 (29). Thereisevidence for a common ancestral origin of human chromosomes 4 and 5(30). These related receptor genes cluster near the centromere on 4q orat the distal half of 5q. Thus, if the progenitor(s) of these genes wereconfined to a single ancestral chromosome, the breakup of linkage mightbe explained by an inversion within the long arm.

The present studies also establish that different PDGF-R genes encodetwo receptor types, with binding properties evidently independent of thecell in which each is expressed. The implications of this observationcan be better appreciated in light of knowledge about other receptorsystems.

There is emerging evidence that as more complex organisms have evolved,mechanisms of intercellular communication have increased in complexityas well. The related EGF and TGF_(α) molecules interact with similaraffinities with a common receptor, the EGF receptor (31). Differentpatterns of developmental and tissue expression of these growth factors(32) presumably account for their present existence.

There are increasing examples of evolutionarily divergent receptor genesas well. The products of such genes can respond to completely differentligands, as is the case of PDGF and CSF-1 receptors (33, 34), or,alternatively, to related ligands, as with the IGF-I and insulinreceptors (35). Here the developmental and tissue specific expression ofboth the receptors and their ligands, as well as the biochemicalresponses triggered, have evolved with the complexity of the organism.

As demonstrated in the present studies, the responses mediated by PDGFnot only involve different dimeric forms of the related ligands encodedby two genes, but two related genes encoding different PDGF receptors aswell. In addition to their differences in tissue specific expression(34, 36), the two PDGF gene products are known to differ in theirrelative secretory capacity. The PDGF-A chain is much more efficientlyreleased than is the B chain (37), giving the former the possibility ofacting at greater distances.

In view of the present evidence of coordinate expression of the two PDGFreceptor genes in all normal tissues so far examined, their tissuespecific expression may not be a major determinant of their functions.However, application of the methods of the present invention to acomprehensive survey of the expression of each receptor type duringembryonic development and in homogeneous normal cell populations mayuncover evidence of differential regulation.

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For purposes of completing the background description and presentdisclosure, each of the published articles, patents and patentapplications heretofore identified in this specification are herebyincorporated by reference into the specification.

The foregoing invention has been described in some detail for purposesof clarity and understanding. It will also be obvious that variouscombinations in form and detail can be made without departing from thescope of the invention.

1. A method of determining the ability of a compound to bind toPlatelet-derived growth factor receptor alpha (α PDGFR) comprising: a)providing a sample of cells having a recombinant construct expressing αPDGFR; b) exposing said sample of cells expressing α PDGFR to a compoundsuspected of binding to α PDGFR; and c) measuring the amount of α PDGFRactivity after exposure of the cell sample.
 2. The method of claim 1,wherein the compound is an agonist of α PDGFR activity.
 3. The method ofclaim 1, wherein the compound is an antagonist of α PDGFR activity. 4.The method of claim 1, wherein the ability of the compound to bind to αPDGFR is compared to the ability of the PDGF AA isoform to bind to αPDGFR.
 5. The method of claim 1, wherein the ability of the compound tobind to α PDGFR is compared to the ability of the PDGF AB isoform tobind to α PDGFR.
 6. The method of claim 1, wherein the ability of thecompound to bind to α PDGFR is compared to the binding ability of anantibody or fragment thereof that binds to α PDGFR but not to β PDGFR.7. The method of claim 1, wherein the cells expressing α PDGFR are acell line expressing α PDGFR.